In vitro DNA manipulation and the attendant transfer of genetic information have developed into a technology that allows the efficient expression of endogenous and foreign proteins in microbial hosts. Recombinant DNA techniques have made possible the selection, amplification and manipulation of expression of proteins and peptides. For example, changes in the sequence of the recombinantly produced proteins or peptides can be accomplished by altering the DNA sequence by techniques like site-directed or deletion mutagenesis.
Some modifications to a recombinantly produced protein or peptide, however, cannot be accomplished by altering the DNA sequence. For example, while the C-terminal .alpha.-carboxyl group in many naturally occurring protein and peptides often exists as an amide, this amide typically is not produced directly through expression. Rather, a precursor protein is produced by expression and the amide is biologically produced in vivo from the precursor protein.
Moreover, although expression of any foreign protein in any microbial host is theoretically possible, the stability of the protein produced often limits such practice and results in a low yield. In particular, small foreign proteins and oligopeptides cannot be overproduced in most cellular hosts. Expression of a small peptide in a host cell raises the possibility that the host will assimilate the peptide. For example, where size of the desired peptide is no more than about 60 to 80 amino acid units in length, degradation usually occurs rather than end product accumulation.
In response to this problem, small peptides have been expressed as part of fusion proteins which include a second larger peptide, such as a marker peptide (e.g., .beta.-galactosidase or chloramphenicol acetyl transferase). While the use of a fusion protein may avoid assimilation, this approach may lead to other problems. Purification is often not very efficient or effective. Many of the marker peptides are such large molecular weight proteins that the desired protein constitutes only a small fraction of the fusion protein.
Another approach involves the expression of a recombinant construct which includes multiple copies of the desired peptide (a multicopy construct). The multicopy construct may or may not include a marker peptide or other leader sequence. Typically, the multicopy construct is designed so that the molecular weight is sufficient to prevent assimilation by the host cell.
The multicopy approach has typically been carried out with methionine residues positioned between the desired peptides. While such constructs can be selectively cleaved with cyanogen bromide, the use of multicopy construct with methionine cleavage sites is limited to the production of product peptides which lack a methionine residue (Met). In addition, cleavage of a multicopy construct at a methionine produces peptides with a C-terminal homoserine (Hse) lactone. This unnatural amino acid residue can be converted to the free acid or amide by ring opening. The amidated peptide, however, contains the unnatural amino acid residue, homoserine, as its C-terminal residue. Thus, known multicopy based methods which make use of methionine as a cleavage site do not permit the production of .alpha.-amidated forms of native peptide sequences.
Other reports of multicopy-based peptide production disclose the use of an acid sensitive cleavage site, -Asp-Pro-, or a tripeptide linker sequence which is cleaved by a specific pair of proteases (trypsin and .alpha.-chymotrypsin). Neither of these methods, however, permits the generation of the .alpha.-amidated form of peptides without placing some limitation on the amino acid sequence of the product peptide.
Accordingly, there is a continuing need for efficient flexible, inexpensive and convenient methods for the recombinant production of C-terminal amidated peptides. In particular, there is a need for methods which permit the production of a recombinant peptide in its .alpha.-amidated form without any limitations as to the amino acid sequence of the peptide. It is therefore an object of the present invention to provide an improved method for the production of a recombinantly produced C-terminal .alpha.-amidated peptide. A further object is to provide a simple and efficient method for modification of a recombinantly produced peptide which permits the exchange of an unnatural C-terminal homoserine residue with the amidated form of another amino acid. These and other objects are accomplished by the present invention.